MHC class I genes are transcriptionally regulated through the promoter and regulatory elements in the 5' flanking sequence, to achieve varying levels of expression in all tissues. The class I promoter contains both a variant TATA box and initiator element, but neither of these elements is critical for basal expression of class I. Rather, we have identified a novel core promoter element that is required for the transcription of MHC class I genes. Screening for DNA-binding factors that are specific for this sequence is in progress; one factor has been identified and is being characterized. The canonical TATA and Inr boxes may function in activated transcription. In vitro transcription assays are being used to further characterize the preinitiation complex. The 5' flanking DNA sequence of a swine class I gene, extending from the promoter to 1100 bp. upstream, contains a series of negative and positive regulatory elements. Basal transcription of class I genes varies among the tissues, as the result of negative regulation mediated largely by a tissue specific regulatory domain consisting of overlapping enhancer and silencer elements, located between -700 and -800 bp. Enhancer binding activity is present in all cell extracts, whereas silencer binding is inversely proportional to the level of class I expression, leading to our proposal that class I genes are negatively regulated. Biochemical characterization of the regulatory factors has demonstrated that each factor consists of at least two distinct components, one of which appears to be common to both factors. Both factors are redox- sensitive. The enhancer factor is about 30 kD; one subunit is glycosylated. The silencer factor is approximately 95 kD. Extensive purification of these factors has been accomplished. Another regulatory element has been identified that responds to activation by the transcription factor USF. Although the brain does not normally express class I, studies in our lab suggest that this is the result of active repression. Further analysis has also revealed the existence of a brain-specific enhancer of class I gene expression.